In accordance with the definition of the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) glycated haemoglobin (HbA1c) is the stable product of a coupling of glucose to the N-terminal valine of the beta chain of the haemoglobin and the amount of HbA1c in relation to the amount of overall haemoglobin (mmol HbA1c per mol total haemoglobin) is representative of the average blood glucose level of the last eight weeks before the blood sample was taken and is therefore also referred to as “long-term blood sugar”. The HbA1c value which should normally be in the range of 20 to 42 mmol/mol is therefore an important indicator for the diagnosis and treatment of diabetes mellitus.
Known methods of determining HbA1c are for example immunoassays, for example turbidimetric immunoassays (TIA) and high power liquid chromatography (HPLC). An enzymatic method has also been available for some time, in which a reaction of glycated haemoglobin with a fructosyl amino acid oxidoreductase (FAOD) is quantified.
In the enzymatic investigation—as moreover is also the case with all other HbA1c methods—the first step is to haemolytically rupture the erythrocytes in the blood sample to release the HbA1c contained therein. The released glycated haemoglobin is then brought into contact with a proteolytic agent to produce glycated haemoglobin degradation products. Those proteolytically produced degradation products include the fructosyl valine (Fru-Val) and fructosyl valine histidine (Fru-Val-His) or even longer-chain functosyl peptides, which are cleaved from the amino-terminal end of the beta chain of glycated haemoglobin. The fructosyl amino acid or fructosyl peptide is oxidised by the activity of the enzyme fructosyl amino acid oxidase (FAOX) or the enzyme fructosyl peptide oxidase (FPOX), wherein a result of that oxidation step is the production of hydrogen peroxide (H2O2).
The amount of hydrogen peroxide produced in the above-mentioned oxidation step correlates with the amount of fructosylated amino acid or peptide. Accordingly the amount of hydrogen peroxide produced in this step is a measurement in respect of the amount of HbA1c in the sample. Therefore, determination of the amount of HbA1c can ultimately be effected by quantifying the amount of hydrogen peroxide, for example on the basis of a colour reaction which is to be evaluated photometrically and which stoichiometrically correlates with the amount of hydrogen peroxide. In principle however it is also possible to correspondingly use any other analysis method for quantifying the amount of hydrogen peroxide in a sample.
In a given method of quantifying hydrogen peroxide, a reduced leuco dye is oxidised with hydrogen peroxide. That method however entails the difficulty that autooxidation of the leuco dye causes a non-specific blank value signal and an increase in the spectral background which causes difficulty in precise photometric measurement of the analyte signal. On the other hand leuco dyes have the advantage over other dyes that they usually have higher molecular absorption coefficients. In addition for the major part leuco dyes have such high absorption maxima that optical influencing by interaction with constituents of the blood like for example bilirubin and haemoglobin can generally be disregarded.